The focus of this proposal is the regulation of the mammalian bumetanide- sensitive Na-K-2Cl cotransporter (rBSC) found in the thick ascending limb of Henle. This protein is the pharmacological receptor for loop diuretics (furosemide, bumetanide and ethacrynic acid), and plays a pivotal role in renal physiology. The sponsor's laboratory has recently cloned and sequenced the first three members of the electroneutral Na-(K)-Cl cotransporter family, which includes rBSC. Phase I of this proposal deals with the role of direct phosphorylation of the rRBC cotransporter protein in the regulation of its activity and ionic stoichiometry. Antibodies will be generated to unique cytoplasmic fragments of rBSC, as expressed in recombinant fusion proteins. These antibodies will be used to characterize the rBSC protein and demonstrate that it is a phosphoprotein. Two dimensional electrophoresis and autoradiography of radiolabeled rBSC peptide fragments will be used to identify residues phosphorylated by PKA and PKC. Phosphopeptides will also be purified by HPLC and sequenced. Phosphorylation sites identified by this protocol will be altered by site- directed mutagenesis of the rBSC cDNA. Stable transfectants of both wild type and mutant rBSC will be created, in cell lines which lack endogenous Na-K-2Cl cotransport. The effect of these defined mutations will be assessed by functional assay of ion cotransport. The transcriptional regulation of the BSC gene is the focus of Phase II of this grant. The effect of PKA and PKC on induction of BSC mRNA will be assessed in Northern blot experiments and nuclear run-off assays, using the rat (rBSC) and mouse (mBSC) cDNA probes. The mouse BSC gene will be cloned from a genomic library. Its intron-exon structure will be determined, and the 5'-promoter region will be cloned and sequenced. Functional characterization of the mBSC promoter will define which elements are required for tissue-specific expression and for the response to PKC. Characterization of nuclear proteins which interact with the mBSC promoter will begin towards the end of the grant period. The long-term goal of Phase II is the identification and cloning of transcription factors which regulate the expression of the BSC gene. By analogy with other tissues, these transcription factor(s) are postulated to play a role in the development, growth and function of the thick ascending limb of Henle.